Microbial sensory rhodopsins, membrane-embedded 7-helix light-sensors widespread among prokaryotes and unicellular eukaryotes, are remarkably diverse in their signaling mechanisms. In archaea and bacteria they mediate phototaxis by protein-protein interaction in membrane-embedded receptor-transducer (SR-Htr) complexes. In contrast, homologous sensory rhodopsins in algae, channelrhodopsins (ChRs), mediate phototaxis by depolarizing the algal plasma membrane. The advantages provided by light activation, namely temporal precision and spectroscopic tools, have made microbial rhodopsins paradigm systems for membrane protein function and for understanding how evolution modifies existing protein scaffolds to create new protein functions. In addition to their basic science interest, sensory rhodopsins have given birth to a new technology, optogenetics, which uses ChRs to control membrane potential in animal cells, enabling light-triggered neuron firing. ChRs have become widely used research tools in neurological disease research and offer promise as therapeutic agents. Our goal is to elucidate the underlying principles of microbial sensory rhodopsin mechanisms at the level of atomic structure/molecular function. We have established that the rhodopsin subunits of SR-Htr complexes, and provide evidence that ChRs as well, share the same light-induced conversion between two conformers with light-driven rhodopsin proton pumps. However, sensory rhodopsins have evolved new chemical processes, not found in their proton pump ancestors, to alter the consequences of the conformational change. Experiments are designed to: 1) determine the first atomic-resolution X-ray crystal structure of the elusive transient light-induced conformer of microbial rhodopsins by exploiting our recent finding of conditions in which this conformer exists as a stable form in the dark in the attractant receptor SRI- HtrI complex; 2) to elucidate in the repellent receptor SRII the interplay between the conformer transition and a steric trigger during photoisomerization of retinal that together comprise the intramolecular pathway of signal transfer to HtrII; 3) to apply our knowledge of SRI and SRII and multidisciplinary tools to the ChRs. The channel activity of ChRs has been detected and investigated exclusively by photoelectric measurements in living algae or animal cells, at concentrations of ChRs not amenable to optical or molecular spectroscopy. We propose to develop an in vitro system for light-gated channel activity of purified ChRs and elucidate the protein's phototransduction mechanism. A final aim follows from our studies of phototaxis in algae which establish that ChR-mediated depolarizing currents are amplified ~1000-fold compared to their activity in heterologous systems, e.g. neurons. We propose a strategy to identify the amplification component(s) and test our hypothesis that non-voltage-gated Ca2+ channels are directly activated by physical interaction with ChRs in the algae. In addition to answering basic mechanistic questions, these experiments have the potential for major impact on optogenetics, enhancing use of this technology in research and enabling therapeutic applications.